Abstract

We have demonstrated that a specific anti-Gli1 shRNA can ablate Gli1 in A2780-CP70 cells, with no effect on Gli2. Upon ablation of Gli1, platinum-DNA adduct repair is inhibited, along with inhibition of ERCC1, XPD, XRCC1, and c-jun. We therefore asked the question of whether there were other subcellular pharmacologic effects, with respect to cisplatin, in these cells. We assessed total cellular accumulation of cisplatin under three conditions: pretreatment with the anti-Gli1 shRNA; pretreatment with a scrambled shRNA control; and pretreatment with Fugene which was used as the transfection agent. We also assessed platinum-DNA adduct levels, at the same time points that total cellular accumulation was measured. All platinum measurements were performed by AAS with Zeeman background correction. In each independent experiment (n=4), we plated cells on day 1; transfected with shRNA 16-18 hours later; treated with cisplatin for 1 hour at 30 μM (the IC50 under these conditions); and, harvested cells at different time points. Those time points were: zero hours (immediately after the 1 hour cisplatin treatment); 6 hours later; or 12 hours later. At zero hours, measured levels of pt-DNA damage were equivalent between the three conditions. At zero hours, measured levels of total cellular platinum were reduced by 40-45% in anti-Gli1 treated cells; suggesting inhibition of cellular accumulation. At 6 hours, scrambled controls showed reductions in total cellular platinum by ∼40%, demonstrating excretion of cisplatin. This is consistent with previously published work (JCI 1991). Cells treated with anti-Gli1 shRNA showed no reductions in total cellular platinum at 6 hrs, nor at 12 hrs; suggesting inhibition of the processes by which cells may eliminate this drug. Cells treated with anti-Gli1 shRNA showed statistically significant inhibition in platinum-DNA adduct repair, as compared to cells treated with scrambled shRNA. Control cells repaired 67.5% of pt-DNA adducts over 12 hours; as compared to 23.7% repair in cells treated with anti-Gli1 shRNA; p2=0.015. We conclude that ablation of Gli1 in cisplatin-resistant A2780-CP70 human ovarian cancer cells, has at least three effects on the subcellular pharmacology of cisplatin. There is inhibition of processes that determine: cellular uptake of drug; cellular elimination of drug; and platinum-DNA adduct repair.